Published December 1, 1985
Human Endothelial Cells are Chemotactic to
Endothelial Cell Growth Factor and Heparin
VICTOR P. TERRANOVA, ROBERTA DsFLORIO, RAYMOND M. LYALL,
SUSANNE HIC, ROBERT FRIESEL, and THOMAS MACIAG
Department of Cell Biology, Revlon Biotechnology Research Center, Rockville, Maryland 20850
Chemotactic behavior is a property of a variety of cell types
engaged in biological processes including inflammation,
wound repair, organ development, neurite outgrowth, and
tumor invasion (29). Factors that modify cellular chemotaxis
are also important as modulators of cellular growth and
differentiation ( 17, 26, 28).' Extracellular matrix proteins such
as fibronectin and laminin have been shown to stimulate
mammalian cell motility in a variety of cell types (1, 25, 28,
39) 2 and may play a prominent role in the process of differentiation (1, 3, 12, 15, 16, 22, 27, 36, 40, 41). 1 Endothelial
cell growth factor (ECGF) 3, the principle polypeptide mitogen
for human endothelial cells in vitro (20), has been implicated
in neovascularization (19, 30). In addition, it has been demonstrated that the glycosaminoglycan, heparin, is a modulator
of the biological activity of ECGF (30, 37). Specifically, heparin increases the activity of the polypeptide mitogen by a
mechanism involving ECGF receptor occupancy (30). Furthermore, mast cell heparin has been shown to stimulate
migration of bovine capillary endothelial cells but not endothelium from bovine aorta (4). Since heparin in combination
Terranova, V. P., M. Aumailley, L. H. Sultan, G. R. Martin, and
H. K. Kleinman, manuscript submitted for publication.
2 Terranova, V. P., R. DiFlorio, E. S. Hujanen, R. M. Lyall, L. A.
Liotta, V. Thorgeirsson, G. P. Siegle, and E. Schiffman, manuscript
submitted for pubication.
3Abbreviation used in this paper: ECGF, endothelial cell growth
factor.
2330
with ECGF promotes cell growth (20, 30, 37) and mediates
alterations in endothelial cell phenotype (37), we examined
their chemotactic properties. We report here that ECGF and
heparin are indeed chemotactic for human umbilical vein
endothelial cells.
MATERIALS AND METHODS
Human Endothelial Cell Cultures: Humanendothelialcellsderived from the umbilical vein were propagated as previously described (21). In
all cases, early passage cells were used (equal to or less than 25 cumulative
population doublings). The cells used for the chemotaxis assays were removed
from the cell culture dishes by incubation in a divalent, cation-free, balanced
salt solution containing 0.1% EDTA, 0.1% EGTA in 25 mM Hepes, l0 mM
Na2 HCO3, 6 mM K2HPO4, 100 mM NaCl and 60 mM mannitol, pH 7.4.
Chemotaxis Assay: Chemotaxis was assayed in the modified Boyden
chamber as previously described (29). Briefly, human endothelial cells were
suspended at a concentration of 2.5 x l05 cells/ml in Medium 199 containing
200 ug/ml bovine serum albumin and placed in the upper compartment of the
Boyden chamber. The lower compartment, which contained the chemoattractant, was separated from the upper compartment by a nuclepore filter (Neuro
Probe, Inc., Cabin John, MD; pore diameter, 8 urn). After a 4-h incubation at
37°C, filters were removed, washed, stained with hematoxylin, and mounted
bottom side up on glass slides. Ten high power fields (× 400) were counted to
determine the number of cells that had migrated entirely across the 100-urn
width oftbe filter. Negative controls were the number of cells that had migrated
in the absence of an attractant, in all cases, the SEM did not exceed 10%.
Preparation of 5ubstrates and Antibodies:
TypeI collagenwas
prepared from lathyritic rat skin (5); type lI collagen from a rat chondrosarcoma
(35); type llI collagen from fetal calf skin (9); type IV collagen from the EHS
tumor ( 18); and type V collagen from human placenta (6). ECGF was prepared
THE JOURNAL OF CELL 81OLOGY • VOLUME 101 DECEMBER 1985 2 3 3 0 - 2 3 3 4
© The Rockefeller University Press • 0021-9525/85,/12/2330]05 $1.00
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ABSTRACT The response of human endothelial cell migration to various extracellular matrix
components and growth factors has been assessed. Human endothelial cells demonstrate
increased chemotaxis and chemokinesis when placed in a modified Boyden chamber with
endothelial cell growth factor (ECGF) used at a concentration of 10-9 M. Anti-ECGF antibody
inhibits the chemotactic response. Heparin (10 -a to 10-l° M) was also chemotactic and was
shown to potentiate the chemotactic activity of ECGF. Although laminin, fibronectin, the
polypeptide (epidermal, fibroblast, and nerve) growth factors, and collagen types I, II, Iit, IV,
and V demonstrate a chemotactic response, these activities were one third to one half less
than observed with ECGF. These data suggest that ECGF and heparin may play a significant
role as response modifiers of human endothelial cell migration which may be relevant to
tumor metastasis, wound healing, and atherogenesis.
Published December 1, 1985
as previously described (7, 21). Briefly, bovine brains were extracted at neutral
pH in a low ionic strength buffer. The extract was next subjected to acid
extraction and a series of ammonium sulfate precipitations. The resulting
material was further purified by heparin-Sepharose affinity chromatography
followed by reversed-phase high performance liquid chromatography. This
material yields a single band on SDS PAGE, a single UV absorbing peak on
reversed-phase high performance liquid chromatography, and a single NH2terminal residue as determined by automated Edman degradation. Laminin
and fibronectin were purified as previously described (14, 18, 38), and antibodies to laminin and fibronectin were raised in rabbits (10). Their specificities
were confirmed by immunoprecipitation and Western blot analysis. Antibody
to purified ECGF was prepared as previously described (20). The polypeptide
(epidermal, fibroblast, and nerve) growth factors were purchased from Collaborative Research, Inc. (Lexington, MA). Insulin was obtained from Sigma
Chemical Co. (St. Louis, MO). Heparin was obtained from Armour Pharmaceutical Co. (Kankehee, IL).
RESULTS
ECGF is a Chemoattractant for
Human Endothelial Cells
60
US
I
50
r~
.--I
LU
LL
a:
40
LU
0rt
-I-
30
-T.--I
.~
LU
(..)
20
10
0
0
1040
1@9
10"s
PROTEIN CONCENTRATION
Polypeptide or
Glycoprotein
BSA
EGF
FGF
NGF
Insulin
Collagen I
Collagen II
Collagen Ill
Collagen IV
Collagen V
Laminin
Fibronectin
Number of cells/high power field
I 0 -~° M
10 -9 M
I 0 -8 M
12+5
20 _ 4
13+5
11 ___3
10 4- 2
18 + 4
12 __.4
25 4- 6
25 4- 5
25 4- 6
20 4- 4
21 4- 5
12 + 2
24 ___3
15___5
12 _ 3
10 4- 1
19 __-2
13 __. 3
29 4- 4
21 + 3
27 + 5
22 4- 4
23 4- 4
10+4
20 4- 5
14+5
13 4- 6
8 + 2
22 + 3
11 + 4
20 4- 4
22 4- 5
18 4- 5
25 _ 5
28 4- 6
Chemotactic assays were performed in triplicate as described in Materials
and Methods. All factors were examined at concentrations between 10-8 and
10-1° M.
response of the cells (Fig. 1) relative to either the heparin or
ECGF response. Although laminin and fibronectin stimulated
endothelial cell migration at 10-9 M, this response was only
30 to 40% of the chemotactic response observed with ECGF
and heparin at 10 -9 M (Table I). In some cases ECGF,
heparin, and collagen types III, IV, and V at increased concentrations revealed a decline in chemotaxis (Table I, Fig. I).
This decrease in response to higher concentrations of chemoattractants is commonly observed in assays of this type and
is generally ascribed to either receptor desensitization or the
possibility of a gradient breakdown (29).
The concentration of ECGF in the upper and lower wells
of the Boyden chamber was varied in a systematic fashion to
distinguish chemotaxis from chemokinesis and also to distinguish positive from negative chemotaxis (42). We observed
an increase in endothelial cell chemokinetic motility as a
function of the concentration of ECGF (Fig. 2). We also
observed a substantial chemotactic activity. The number of
cells that had migrated in positive gradients was substantially
greater than those that migrated in negative gradients. These
data demonstrate that ECGF induces a true chemotactic
response in human endothelial cells. An additional checkerboard assay was performed using heparin as the chemoattractant. In this experiment we observed positive chemotaxis and
positive chemokinesis. No movement of human endothelial
cells was noted when a negative gradient was established (Fig.
3).
Antibodies Against ECGF Inhibit
ECGF-induced Chemotaxis
10-7
[MI
FIGURE 1 Chemotactic response of human endothelial cells to
ECGF and heparin. Chemotaxis was assayed in the modified Boyden chamber. After a 4-h incubation at 37°C in a 100% humidified
chamber containing 5% CO2, the filters were removed, fixed in
100% methanol, and stained with hematoxylin and eosin. Because
of the heterogeneous nature of heparin preparations, we assumed
an average molecular mass of 2 x 104 daltons. Molar concentrations
for heparin are based on this assumption. The data are expressed
as the average number o f migrated cells per high power field ( x
400) + one standard deviation. Each assay was performed in triplicate. The number of migrated cells per filter did not differ by more
than 10%.
Human endothelial cells treated with an antibody directed
against ECGF, which inhibits ECGF-induced endothelial cell
proliferation and ECGF receptor occupancy (20), showed
marked inhibition of the chemotactic response to ECGF.
Antibodies against laminin (data not shown) or fibronectin
had no effect on ECGF-induced human endothelial cell migration (Fig. 4). In a series of separate experiments, human
endothelial cells were assayed for their chemotactic response
to the various antibodies. We observed no movement of cells
to either anti-ECGF, anti-laminin, or anti-fibronectin in the
absence of ECGF (Fig. 5). In addition, when ECGF was added
to the lower compartment, a maximal response was observed
TERRANOVA ET AL. Human Endothelial Cells Move to ECGFand Heparin
2331
Downloaded from on June 15, 2017
When human endothelial cells were placed in the upper
compartment of a modified Boyden chamber at a density of
2 x l0 s cells per chamber (42), we observed that ECGF and
heparin, alone or in combination, stimulated human endothelial cell migration (Fig. 1). The optimal concentration of
ECGF for directed cell movement w a s 10 -9 M. Heparin also
stimulated human endothelial cell migration at 10 -9 M. The
addition of both ECGF and heparin increased the chemotactic
Chemotactic Response of Human Endothelial Cells to
Biological Response Modifiers
TABLE I.
Published December 1, 1985
ECGF CONCENTRATION (M) IN UPPER CHAMBER
uJ
a3
~
~<~
~-~
[
]
I 0
I
:~
Z
0
~
10 10
.
I ~
I 12 + 4
I
~
10 + 6
.
I
.^ I
~
I 10-'0 ] 26 5- 9
18 + 4
.
51 + I~
10-9
.
.
.
10 .8
.
9 + 8
.
10-7
13 + 7
12 + 4
19 _+ 10
9 + 5
17 5- 9
~
30 + 6 .
~
.
21 + 6
.
17 + 4
FIGURE 2 Checkerboard analysis of the motile response of human endothelial cells to ECGF. Chemotaxis assays are performed as described in Materials and Methods. Each assay was performed in
.
triplicate. Numbers within the inner box express
the number of migrated cells per high power field
I
400 The concentration of EC E was var,ed
Z
O
[ .^ a
I tU-~
[
I .
[ ~
I
.
.
5- 12
I .^_,
I . . . . . . . . . .
2
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I 10-7
I
I 31 5- 7
t l ~ 5- ],J
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.
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7
-
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~
. . . .
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- ~.
33 + 15
~
48 + 11
the upper and lower chambers as indicated. The
response of human endothelial cells to the absence of a gradient (chemokinesis)are shown on
the diagonal and to a negative gradient above the
-
4
-
dmgonal. Response to a positive gradient (chemo-
taxis) is shown below the diagonal.
"17 + 6
§
HEPARIN CONCENTRATION (M)
IN UPPER CHAMBER
I 0
I"'~
3
~0±3
9~
~2:~
~3
I
~
~'"
<:IO
" ' t iI-- ~<~
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19±5
~
15 ± 6
-I-4
11±4
O
O.
88__+2
+ 2 10±3
10+3
~Z CC
\1~
T
"I-
BSA
~r ::]:
7"
Anti-Fibroneetin
3o
"1"
CO
"'O
o~
I
-~
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z
50 /
L ,
110-*I 3s±4
Ie±4
I
I_I
L~
2o'±4
1o±2
\
30±5
25±4
24±4
I
co
12±31
I
23±6
\
27±5J
20
I
\
Anti-ECGF
,oL
oLd,,
0
10-6
,
10-5
,
-:
10 -4
10 -3
10 "2
",,1
FIGURE 3 Checkerboard analysis of the motile response of human
endothelial cells to heparin. Chemotaxis assays are performed as
described in Materials and Methods. Each assay was performed in
triplicate. Numbers within the inner box express the number of
migrated cells per high power field (x 400). Heparin concentrations
are varied as indicated. The response of human endothelial cells to
the absence of a gradient (chemokinesis) are shown on the diagonal
and to a negative gradient above the diagonal. Response to a
positive gradient (chemotaxis) is shown below the diagonal.
(Fig. 5). Furthermore, anti-ECGF antibody did not inhibit
endothelial cell movement to either laminin or fibronectin
(data not shown). These studies demonstrate the specificity of
ECGF-induced endothelial cell migration and further suggest
that the inhibition of cell motility by anti-ECGF was not due
to a nonspecific antibody interaction with human endothelial
cells.
Chemotactic Activity of Other Biological
Response Modifiers on Human Endothelial Cells
Additional factors which, in general, modulate mammalian
cell proliferation and differentiation were examined for their
2332
THE JOURNAL OF CELL BIOLOGY • VOLUME 101, 1985
ANTIBODY DILUTION
FIGURE 4 Inhibition of ECGF-induced endothelial cell chemotaxis.
Chemotactic response of human endothelial cells to ECGF was
measured in the presence and absence of antibodies. Anti-fibronectin (initial concentration of 100 ~g/ml protein) and anti-ECGF
(initial concentration of 100 #g/ml protein (20) were incubated with
human endothelial cells in the upper compartment of the Boyden
chamber. ECGF (10-9 M) was used as the chemoattractant in the
lower well of the Boyden chamber. The chemotaxis assay was
performed as described in Materials and Methods.
chemotactic activity with human endothelial cells. These included epidermal growth factor, basic pI-fibroblast growth
factor, nerve growth factor, insulin, and collagen types I, II,
III, IV, and V. The various factors were examined for chemotaetic activity in the modified Boyden chamber at concentrations of 10-8, 1 0 -9, and 10-l° M; the results obtained are
shown in Table I. Collagen types I, III, IV, and V and EGF
were capable of stimulating one third to one half maximal
stimulatory capacity as compared to ECGF. Collagen type II
was without effect in chemotactic stimulation. Both fibroblast
and nerve growth factor were also without effect. These data
Published December 1, 1985
o
.J
uJ
,.-r 5 0
l=
ILl
,PLUS 10-9 M ECGF
on 4 0
',r
~
(~ ANTI FIBRONECTIN
3o _
.J
_J
°
20
m
10
u.
o
/~ ANTI LAMININ
[]
ANTI E C G F
D
Z
o -//
0
L
I
10-5
t
10-4
10-3
I
I
10-2
10-1
A N T I B O D Y DILUTION
TABLE II. Chemotactic Response of Human Endothelial Cells to
Various Biological Response Modifiers with and without 10 -9 M
Heparin
Polypeptide or
Glycoprotein
EGF
FGF
Laminin
Fibronectin
Number of cells/high power field
+ Heparin
26
14
25
25
+
+
+
3
3
3
2
- Heparin
25
14
22
23
+ 4
+ 4
+ 2
_+ 3
Endothelial cell chemotaxis was assayed using 10-9 M polypeptide or glycoprotein with and without the addition of 10-9 M heparin. Results are the
mean of four experiments plus and minus one standard deviation.
demonstrate that ECGF is a more potent chemoattractant for
human endothelial cells in vitro relative to other known
chemoattractants. In addition, we tested the ability ofheparin
in combination with epidermal growth factor, fibroblast
growth factor, laminin, and fibronectin to stimulate directed
movement of endothelial cells. We observed no increase (or
decrease) in chemotactic responsiveness when heparin (at 10 -9
M) was included (Table II).
DISCUSSION
These data demonstrate that human endothelial cells exhibit
a chemotactic response to both ECGF and heparin. Since the
chemotactic activity of ECGF is potentiated by heparin at
suboptimal concentrations, these data suggest an interaction
between the polypeptide and the glycosaminoglycan. This
proposed interaction is consistent with the ability of heparin
to bind ECGF (20) and modulate the mitogen activity of the
polypeptide (30, 37). Previous studies have demonstrated that
the modulation by heparin occurs at the receptor level (30).
The interaction between ECGF and heparin increases the
dissociation constant for ECGF receptor occupancy and may
We thank Drs. W. Terry and C. Smith for their support and critical
comments.
This work was supported in part by grants AG84807 and
HL310765 to T. Maciang from the National Institutes of Health.
Received for publication 10 July 1985, and in revised form 28 August
1985,
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TERRANOVAET AL. Human Endothelial Cells Move to ECGFand Heparin
2333
Downloaded from on June 15, 2017
FIGURE 5 Antibodies against fibronectin, laminin, and ECGF are
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2334
THE JOURNAL OF CELL BIOLOGY . VOLUME 101, 1985
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